The long-term goal of this project is to understand how the Hox family of homeodomain transcription factors regulate their target genes in vivo. Hox proteins are critical regulators of animal development. They also underlie many human diseases. Prior work suggests that these DNA binding proteins use at least two cofactors, Extradenticle and Homothorax, to select the correct sets of target genes in vivo. Extradenticle and Homothorax are also homeodomain proteins and are also implicated in various leukemias. Once bound to DNA, additional factors are then thought to be recruited to Hox/Extradenticle/Homothorax complexes to control target gene expression. These complexes can be used for both gene activation and gene repression. Using Drosophila melanogaster as the experimental system, the experiments proposed here are in part aimed at identifying these additional factors. Some of these are expected to be DNA binding proteins whereas others are expected to be co-repressors or co-activators or proteins that have the capacity to recruit these transcriptional regulators. One set of experiments focus on the Hox-repressed target gene Distalless and are aimed at testing the role of two proteins, recently identified in preliminary experiments, that appear to collaborate with Hox/Extradenticle/Homothorax complexes to repress transcription. A second set of experiments propose to use a biochemical approach to identify and ultimately characterize factors used by Hox/Extradenticle/Homothorax complexes to effect gene regulation. A third aim addresses how Hox proteins achieve DNA-binding specificity in cells where Extradenticle and Homothorax are not available to act as cofactors. Other proteins are hypothesized to act as Hox cofactors in these cells, and experiments are designed to identify and test the function of these putative cofactors in vivo. [unreadable] [unreadable]